The long-term goals of this research are to understand how the herpes simplex virus major DNA-binding proteins, ICP8, moves into and within the cell nucleus and interacts with specific structures and molecules to promote viral DNA replication. This proposal focuses on the mapping of ICP8 domains for DNA- binding, nuclear localization and nuclear retention; the characterization of interactions of ICP8 with the sites of the host cell DNA synthesis; the role of ICP8 in defining the intranuclear location of parental and progeny DNA: and the identification of viral gene products needed to form replication compartments in transfected cells. The DNA-binding region on ICP8 will be identified by sequencing of ts mutant genes that encode ICP8 molecules that localize normally but are thermolabile for DNA binding. Linker insertions and oligonucleotide-directed mutations will also be constructed to identify portions of ICP8 needed for DNA-binding. To map nuclear localization signals, protein fusions will be made to identify the minimal portion of ICP8 needed to target pyruvate kinase to the cell nucleus. Putative nuclear retention signals will be mapped by mutagenesis of a truncated form of ICP8 that appears to be able to diffuse into and accumulate in the nucleus. To probe the function of cell proteins at the prereplicative sites, monoclonal antibodies will be prepared to the ICP8-cell protein complexes. The 3-dimensional distribution of ICP8 in the prereplicative sites will be defined by analysis of the fluorescent image at different focal depths. The role of ICP8 in inhibition of host cell DNA synthesis will be defined in infected and transfected cells. In situ hybridization using biotinylated probes will be used to localize viral DNA and examine the role of ICP8 in defining the nuclear distribution of viral DNA. Using transient expression assays, the minimal number of viral genes needed for assembly of replication compartments will be determined. This will identify any gene products specifically involved in assembly of these structures. These studies may provide information about the organization of the cell nucleus for viral and cellular DNA synthesis. The studies may provide a molecular explanation of how a virus takes over the cell nucleus for replication of its own genome. Thus, they may provide some insight into mechanisms controlling cell DNA replication.